This invention relates to the development of a novel test model and assay for screening the activity of drugs for central nervous system (CNS). More particularly, the invention identifies the use of the fruit fly as a model to test the neuroactivity of test drugs/samples.
Drugs that stimulate or depress CNS play an important role in human therapeutics. They act as anesthetics, analgesics, sedatives, psychostimulants, analeptics, antidepressants, anticonvulsants etc. and are used in the treatment of conditions such as narcolepsy, depression, hyperactivity disorders, epilepsy and drug addiction in human (Wood-Smith and Stewart, 1964, in Drugs in anesthetic practice, Butterworth; Beckman, 1958, in Drugs, their nature, action and use, W. B. Saunder, Green and Levy, 1976, Drug misuse, human abuse, Dekker). Undesirable side effects and ineffectiveness of currently available CNS stimulants/depressants in many situations call for development of novel drugs.
Animal models currently used in neuroactive drug screening mostly include higher animals such as nonhuman primates and rodents. Although their indispensability in advanced stages of drug development can not be denied, using these models in primary rug screening is time, cost and labor intensive. Many components of neuronal signaling are conserved between the fruit fly Drosophila melanogaster and human (Rubin e al, 2000, Science 287:2204-2215; Littleton and Ganetzky, 2000, Neuron 26:35-43). Moreover, many CNS stimulant/depressant drugs used in human therapy also exhibit their neuroactivity in fly (Shaw et al, 2000, Science 287:1834-1837; Hendricks et al, 2000, Neuron 25:129-138; Andretic et al, 1999, Science 285:1066-1068; Li et al, 2000, Curr. Biol. 10:211-214; Baintaon et al, 2000, Curr. Biol. 10:187-194; McClung and Hirsh, 1999, Curr. Biol. 9:853-860). Considering these, the applicants thought of evaluating of potential of Drosophila to serve as a simple, rapid and inexpensive model for screening of CNS active agents.
A variety of CNS stimulant/depressant drugs have originated from plants (Wood-Smith and Stewart, 1964, in Drugs in anesthetic practice, Butterworth; Beckman, 1958, in Drugs, their nature, action and use, W. B. Saunder; Green and Levy, 1976, Drug misuse, human abuse, Dekker, Plotkin, 2000, in Medicine quist in search of nature""s healing secrets, Viking; Gratzer, 2000, Nature 406:235-236). There are several reasons why neuroactive compounds made by plants work on receptors in human brain is also (Lam et al, 1998, Nature 396:125-126). Keeping the above in view, the applicant used the fly model plant samples and developed a simple assay for screening of substances and evaluate their utility in drug screening.
The main object of the invention is to provide a novel method for screening of neuroactive drugs using the fruit fly Drosophila melanogaster as an in vivo model.
Another object is to develop a method for simultaneous screening of antiepileptic and central nervous system stimulant/depressant classes of drugs.
Yet another object is to provide a simple and cost-effective method for studying the neuroactivity of a substance using Drosophila melanogaster as a model.
Accordingly, the invention provides a novel method for screening of neuroactive drugs using Drosophila melanogaster as an in vivo test model. The invention also provides methods for studying the neuroactivity of a substance using Drosophila melanogaster as a model.
The invention relates to a method for screening of neuroactive drugs using the fruit fly Drosophila melanogaster, which comprises the steps of:
a) generating a double mutant line of K+ channel genes in Drosophila melanogaster; 
b) culturing the Sh5eag1 mutant flies on Dorsophila medium under standard conditions; and
c) anesthetizing flies with diethyl ether and observing the time taken by flies to recover from anesthesia.
In an embodiment, decreased arousal time in flies treated with normal fly food mixed with the agent being screened, compared to flies fed on normal fly food, is indicative of analeptic activity of the agent.
In another embodiment, an early arousal in flies treated with normal fly food mixed with both the agent being screened as well as the drug phenobarbital, compared to flies fed on normal fly food mixed with phenobarbital alone, is indicative of an analeptic activity of the agent.
In yet another embodiment, spontaneous locomotor activity is observed.
In still another embodiment, increased locomotor activity in flies treated with normal fly food mixed with both the agent being screened as well as ethanol, compared to flies fed on normal fly food mixed with ethanol alone, is indicative of an analeptic activity of the agent.
In an embodiment, an increased locomotor activity in flies treated with normal fly food mixed with the agent being screened, compared to flies fed on normal fly food, is indicative of a psychostimulant activity of the agent.
Thus, the present invention relates to a novel method for screening of CNS active agents. Ether-sensitive leg shaking phenotype of Drosophila melanogaster K+ channel double mutant Sh5eag1 has been earlier evaluated by the applicants as a target for antiepileptic drug screening (Sharma and Kumar, 2000, U.S. patent application Ser. No. 09/535,517). To further enhance the usefulness of Sh5eag1 in neuroactive drug screening the applicants studied the effect of a CNS depressant drug, phenobarbital, on time taken by flies to recover from ether anesthesia. The drug was found to delay recovery. This suggested that a change in recovery time could be exploited as a screening criterion for CNS active drugs. A comparison between Sh5eag1 and Oregon-R wild-type flies revealed that recovery is faster in the former. This indicated the advantage of mutant over wild-type in speeding up the process of drug screening. The position of Sh5eag1 as an efficient neuroactive drug testing model was therefore further consolidated. The model thus evolved was applied for screening of the targeted activities in plant extracts. Out of 50 plant species screened, one was found to test positive. Further experiments confirmed that the plant substance screened has analeptic and psychostimulant properties (Sharma et al, a substance from Acorus calamus plant with analeptic and psychostimulant properties, a separate patent filed). This demonstrates the practical usefulness of the fly model developed. Accordingly, the present invention provides a CNS drug screening method, which comprises use of the fruit fly Drosophila melanogaster as a whole organism in vivo model for drug testing.
Vertebrate animal models currently used for screening of neuroactive agents are time, labor and cost intensive. The applicants therefore thought of using fruit fly Drosophila melanogaster as a simple, rapid and inexpensive in vivo whole organism behavioral model for primary drug screening. In this context, we previously validated the ether-sensitive leg shaking phenotype in K+ channel double mutant Sh5eag1 as a target for antiepileptic drug screening (Sharma and Kumar, 2000, U.S. patent application Ser. No. 09/535,517). To add value to this model, it was desirous to exploit it for simultaneous screening of other CNS active drugs. Anesthetics produce a profound depression of CNS and, therefore, we wondered if recovery from anesthesia in flies could serve as a simple means to rapidly screen CNS depressants/stimulants. To explore this possibility, the applicants studied the effect of known CNS depressant, phenobarbital, on time taken by Sh5eag1 flies to recover from ether anesthesia. It turned out that the drug delays recovery. A change in recovery time was therefore validated as a criterion for screening CNS active agents. While applying Sh5eag1 in antiepileptic drug screening, the applicants noticed that the mutant flies recover from ether anesthesia earlier than wild-type ones. If it is so, we thought, then the time required to test a sample in the mutant would be less than that in the wild-type and, therefore, screening in Sh5eag1 would be much faster. To verify the difference in recovery time, we compared the performance of Sh5eag1 and the wild-type Oregon-R after ether anesthesia. A quick recovery in the mutant was confirmed. The above mentioned results therefore led to the development of Sh5eag1 as a model for screening CNS depressants/stimulants. This added to the usefulness of the mutant in drug screening because it is already serving as a screen for antiepileptic drugs. The use of Drosophila Sh5eag1 as a test model is novel of illustrated by the following examples, which should not be construed to limit the scope of the invention in any manner.